András Hrabák

The inhibitory effect of nitrite, a stable product of nitric oxide (NO) formation, on arginase

Macrophages contain arginase and an inducible NO synthase, demonstrated by using l‐arginine, the common substrate, for production of both nitric oxide and urea. Arginase was inhibited by nitrite, the stable end product of NO. This inhibition was non‐competitive, and could not be explained by the reaction of nitrite with arginine, or by the irreversible covalent modification of arginase, or by the removal of Mn2+, a cofactor of arginase.

Arginine supply for nitric oxide synthesis and arginase is mainly exogenous in elicited murine and rat macrophages

L-arginine, the precursor of nitric oxide(NO) is provided mainly by extracellular sources in casein-elicited murine and rat peritoneal macrophages. Free extracellular L-arginine(Arg), esters, peptides and proteins containing Arg are the best sources in accordance with the fact that proteolytic activity is high in peritoneal macrophages. The recycling of Arg from citrulline(Cit) was observed but at a low rate. This situation is different from that in endothelial cells where half of Arg is recycled from citrulline. No significant anaplerotic reaction from glutamic acid(Glu) can be demonstrated in peritoneal macrophages.

Indomethacin prevents the induction of inducible nitric oxide synthase in murine peritoneal macrophages and decreases their nitric oxide production

Indomethacin (0.14-.5 mM concentration) inhibits nitric oxide production in murine peritoneal macrophages. This was evidenced by measuring both nitrite production or 14C-L-citrulline formation. The inhibition was caused by the diminution of de novo inducible nitric oxide synthase production as demonstrated by Western blotting experiment. The effect of indomethacin after 4 h treatment was irreversible. NO synthase and arginase activities and the uptake of arginine were not directly affected by the drug. Indomethacin also decreased uridine incorporation in macrophages. The effect of indomethacin on the induction of other enzymes (i.e. arginase) was weaker.

Computer-aided comparison of the inhibition of arginase and nitric oxide synthase in macrophages by amino acids not related to arginine

Macrophages contain arginase and an inducible nitric oxide (NO) synthase that use the same substrate, L-arginine, to produce nitric oxide and urea, respectively. Arginase was inhibited by various amino acids not related to L-arginine. These compounds were bound to the substrate binding site of the enzyme as supported by kinetic studies. Five binding sites were defined in this area by computer-aided analysis, and three complementary sites in a compound were sufficient to give an inhibitory character. NO synthase could not be inhibited by these compounds, but certain derivatives (e.g., putrescine or L-valinol) caused a marked and probably allosteric inhibition. The possible biological importance of these inhibitions in the tumoricid function of macrophages is discussed.

Action of chloroquine on nitric oxide production and parasite killing by macrophages.

Chloroquine is known to inhibit several functions of macrophages, but its effect on the nitric oxide (NO)-dependent parasite killing capacity of macrophages has not been documented. NO synthesis by interferon-gamma-induced mouse and casein-elicited rat macrophages was significantly and irreversibly inhibited by chloroquine. The activity of the inducible NO synthase was not directly altered, but previous incubation of macrophages with chloroquine decreased it. Chloroquine did not alter arginase activity or arginine uptake. NADPH diaphorase activity, an indicator of NO synthase was impaired. Western blotting showed that inducible NO synthase synthesis was blocked by chloroquine. The blocking of NO formation by chloroquine resulted in increased infection of mouse peritoneal macrophages by Trypanosoma cruzi (T. cruzi). This suggests that chloroquine decreases NO formation by macrophages by inhibiting the induction of NO synthase. The findings are further evidence that NO is involved in the anti-parasitic response of macrophages.

Contribution of the heme oxygenase pathway to the maintenance of the hypothalamic blood flow during diminished nitric oxide synthesis

The cerebrovascular effects of the heme oxygenase–carbon monoxide pathway were studied in the rat hypothalamus. Intraperitoneal administration of the heme oxygenase inhibitor zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG, 45 μmol/kg) had no significant effect on the resting cerebral blood flow, but increased hypothalamic nitric oxide synthase activity by 67% without changing the CSF cyclic GMP concentration. After pharmacologic inhibition of nitric oxide synthase, the diminished cerebral blood flow was further reduced by 22% after administration of ZnDPBG, and the effect showed direct correlation with the baseline perfusion level. Therefore, endogenous carbon monoxide may significantly contribute to the cerebral vasoregulation under resting conditions and in pathophysiologic states associated with diminished nitric oxide synthesis.

Adaptation of the hypothalamic blood flow to chronic nitric oxide deficiency is independent of vasodilator prostanoids.

The aim of our study was to investigate the adaptation of the hypothalamic circulation to chronic nitric oxide (NO) deficiency in rats. Hypothalamic blood flow (HBF) remained unaltered during chronic oral administration of the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 1 mg/ml drinking water) although acute NOS blockade by intravenous l-NAME injection (50 mg/kg) induced a dramatic HBF decrease. In chronically NOS blocked animals, however, acute l-NAME administration failed to influence the HBF. Reversal of chronic NOS blockade by intravenous l-arginine infusion evoked significant hypothalamic hyperemia suggesting the appearance of a compensatory vasodilator mechanism in the absence of NO. In order to clarify the potential involvement of vasodilator prostanoids in this adaptation, cyclooxygenase (COX) mRNA and protein levels were determined in the hypothalamus, but none of the known isoenzymes (COX-1, COX-2, COX-3) showed upregulation after chronic NOS blockade. Furthermore, levels of vasodilator prostanoid (PGI(2), PGE(2) and PGD(2)) metabolites were also not elevated. Interestingly, however, hypothalamic levels of vasoconstrictor prostanoids (TXA(2) and PGF(2alpha)) decreased after chronic NOS blockade. COX inhibition by indomethacin but not by diclofenac decreased the HBF in control animals. However, neither indomethacin nor diclofenac induced an altered HBF-response after chronic l-NAME treatment. Although urinary excretion of PGI(2) and PGE(2) metabolites markedly increased during chronic NOS blockade, indicating COX activation in the systemic circulation, we conclude that the adaptation of the hypothalamic circulation to the reduction of NO synthesis is independent of vasodilator prostanoids. Reduced release of vasoconstrictor prostanoids, however, may contribute to the normalization of HBF after chronic loss of NO.

The effect of various inflammatory agents on the alternative metabolic pathways of arginine in mouse and rat macrophages

Abstract.Objective and design: The effects of various inflammatory stimuli on the alternative arginine metabolic pathways in mouse and rat peritoneal macrophages were investigated in vitro and compared. Treatments: Mice and rats were injected i. p. with thioglycollate, carrageenan, casein, BCG and Newcastle Disease Virus (NDV) vaccines. Methods: Peritoneal macrophages were isolated from untreated and treated animals. The activities of nitric oxide synthase (NOS) II and arginase were measured and expressions were followed by Western blotting. The uptake of arginine and nitrite formation of macrophages were also measured. Results: Inflammatory stimuli increased the NO production and the expression and activity of both NOS II and arginase in mice in vitro. On the contrary, the same treatments changed the expression and activity of NOS II only, but not those of arginase in rats. The most marked effects on NO metabolism were produced by casein and NDV treatments. Conclusions: The activity and expression of NOS II and arginase can be stimulated in peritoneal macrophages in vitro by injecting inflammatory agents into the peritoneal cavity. A markedly different response in arginine metabolism was observed in mouse and rat macrophages. Casein treatment was a potent inducer for both enzymes. NDV vaccines induced mainly NOS II, while thioglycollate induced arginase.

Plasma concentration of myeloperoxidase enzyme in pre- and post-climacterial people: Related superoxide anion generation

Neutrophil granulocytes are involved in the pathogenesis of atherosclerosis also through their free radical generation. The aim of the study was to test how extracellular levels of myeloperoxidase (MPO; a granulocyte enzyme playing role in free radical production) change by age and what effect this change has on the production of the free radical superoxide anion by neutrophils. We also wanted to examine whether the antioxidant effect of different steroid hormones is realized through the MPO. Plasma myeloperoxidase concentrations of healthy blood donors were quantified by ELISA. Superoxide anion production was measured by photometry. Myeloperoxidase concentration was significantly lower in plasmas obtained from older women and men than in those from younger subjects. Adding the MPO inhibitors 4-aminobenzoic acid hydrazide (ABAH) and indomethacin to the granulocytes, the generation of superoxide anion increased and the decreasing effect of the steroids on superoxide production was inhibited. Incubating the neutrophils with the product of the reaction catalyzed by MPO itself (hypochlorite anion), we found significant decrease in superoxide generation. According to our results MPO seems to diminish the production of superoxide anion and so probably has an antioxidant ability. Therefore, its lower plasma levels may contribute to the increasing incidence of atherosclerosis and other free radical mediated disorders in old people. Thus, after further studies MPO might become one of the indicators of cardiovascular risk and the scavenger capacity in general.

Enhanced utilization and altered metabolism of arginine in inflammatory macrophages caused by raised nitric oxide synthesis.

Nitric oxide (NO) production was increased in macrophages during inflammation. Casein-elicitation of rodents causing a peritoneal inflammation offered a good model to study alterations in the metabolism of L-arginine, the precursor of NO synthesis. The utilization of L-arginine for NO production, arginase pathway and protein synthesis were studied by radioactive labeling and chromatographic separation. The expression of NO synthase and arginase was studied by Western blotting.Rat macrophages utilized more arginine than mouse macrophages (228+/-27 versus 71+/-12.8pmol per 10(6) macrophages). Arginine incorporation into proteins was low in both species (<15% of labeling). When NO synthesis was blocked, arginine was utilized at a lower general rate, but L-ornithine formation did not increase. The expression of enzymes utilizing arginine increased. NO production was raised mainly in rats (1162+/-84pmol citrulline per 10(6) cells) while in mice both arginase and NO synthase were active in elicited macrophages (677+/-85pmol ornithine and 456+/-48pmol citrulline per 10(6) cells). We concluded, that inflammation induced enhanced L-arginine utilization in rodent macrophages. The expressions and the activities of arginase and NO synthase as well as NO formation were increased in elicited macrophages. Specific blocking of NO synthesis did not result in the enhanced effectivity of the arginase pathway, rather was manifested in a general lower rate of arginine utilization. Different rodent species reacted differently to inflammation: in rats, high NO increase was found exclusively, while in mice the activation of the arginase pathway was also important.